Dear Bomba I am bit confused , Are you running bowtie ---> tophat ----> Cufflink For bowtie run you are using reference genome annotation  in gff3 format  I am curremntly totally unable to figure it out correctly, how i should analyse my RNA seq data Best Ateeq   ________________________________ From: Noa Sher <noa.sher(a)gmail.com&gt; To: Bomba Dam <bomba.dam(a)visva-bharati.ac.in&gt; Cc: galaxy-user(a)lists.bx.psu.edu Sent: Wednesday, February 22, 2012 7:28 AM Subject: Re: [galaxy-user] Cufflinks related problem Hi Bomba I cant know for sure without seeing your files but I originally had the same problem and it ended up being because the way the genome was named in the fasta genome file was not the same as the way it was named in column 1 of my gtf file. I would check that first.  Also- with bacteria you dont want to run cufflinks with default parameters- use the galaxy browser to check how your data looks after tophat and you will most likely see very strange gene-spanning introns. Change the max intron size to 1000-1500 and the min distnace to 101-5nt and you should get results that make much more sense. Good luck Noa On 22/02/2012 00:12, Bomba Dam wrote: Dear Noa, mapping bacterial transcripts using cuffkins. I have kept the default parameters as in Cufflinks and used my custom genome annotation  in gff3 format. The cufflinks seems to work Ok but all the FPKM values in these files are zero. As suggested by other users I have checked the correctness of my GFF3 files. The corresponding fasta file was used for mapping the transcripts using Bowtie. Are there any special trick for mapping bacterial transcriptome. ___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org.  Please keep all replies on the list by using "reply all" in your mail client.  For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:   http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at:   http://lists.bx.psu.edu/